The hypothesis we will test in this proposal is that miRNAs dampen the gap-phase checkpoints in human embryonic stem cells (hESCs) through their mRNA targets, regulators at the gap- phase checkpoints. Previously, our lab showed that miRNA pathway is required for proper cell cycle control in Drosophila germline stem cells. To date, the role of miRNAs in human embryonic stem cell (hESC) cell cycle remains undefined. The goal of this proposal is to determine how miRNAs regulate hESC division, in particular, to identify the critical miRNAs and their cell cycle targets in this cell type. Towards reaching this goal, our preliminary data showed that hESCs lacking mature miRNAs due to knockdown (KD) of Dicer or Drosha have reduced cell cycle kinetics and delayed at both Gap-phases G1 and G2. We also identified 19 candidate miRNAs that may function in hESC division based on their differential expression in undifferentiated versus early-differentiated hESCs using three different hESC lines and large-scale miRNA profiling. In this proposal, in order to identify the critical miRNAs for hESC division (Aim 1), we will setup a rescue experiment using the Dicer KD hESC line as a tool to see which candidate miRNA(s) is sufficient to rescue Dicer KD division phenotype when overexpressed. Loss-of-function analysis will also be performed for the candidate miRNA(s) to determine if it is required for hESC division. Using this strategy, our preliminary studies have tested four out of the 19 candidate miRNAs and showed that miR-195 regulates hESC division. The fact that miR-195 only partially rescues Dicer KD division phenotype and only rescues G2 delay make us believe that more than one miRNAs are involved in hESC cell cycle regulation. In Aim2, we will use miR-195 as an example to study how miRNA regulates hESC cell cycle. We will test if miR-195 predicted targets in cell cycle regulation are responsive to miR-195 levels and whether these targets are utilized in hESCs. Our preliminary data showed that miR-195 directly targets the G2/M checkpoint inhibitory kinase WEE1. We will further test if this has biologically meaning in hESCs by overexpression of WEE1 to see if G2 delay can be reproduced in hESCs. Another predicted target for miR-195, also a negative cell cycle regulator, is CHK1. We will test if CHK1 is a real miR-195 target and whether CHK1 is utilized to control hESC cell cycle. Relevance: This study has a high likelihood to result in finding critical miRNA(s) that regulate cell cycle in hESCs and better understanding on how hESC cell cycle is regulated. This allows better manipulations of hESCs, particular interests in regenerative medicine.